POS0426 PKM2 PROMOTES PRO-INFLAMMATORY MACROPHAGE ACTIVATION IN ANKYLOSING SPONDYLITIS
Authors: Y. Jiang, W. Weng, Y. Liang, S. Zhong, Y. Pan, J. Gu
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Macrophages are vital effector cells in ankylosing spondylitis (AS), leading to autoimmune tissue inflammation through varied effector functions [1]. The pro-inflammation potential of macrophages depends on their metabolic environment. Macrophages also work as principal regulators in T lymphocyte activation. To meet amplified bioenergetic and biosynthetic demands, metabolic pathways are joined to facilitate the proliferation and effector molecule production. A critical question is whether glycolysis and the rate-limiting enzyme, pyruvate kinase isoenzyme M2 (PKM2) could positively influence AS macrophages' inflammatory function. <h3>Objectives</h3> The objectives of the study was to investigate the effect of glycolysis and PKM2 on AS macrophage functions featured by the secretion of inflammatory cytokines and the presentations of co-stimulatory signals. <h3>Methods</h3> Peripheral blood mononuclear cells (PBMC) were isolated from AS patients and differentiated into macrophages by M-CSF. Macrophages were further differentiated into M1 or M2 macrophages by stimulation with IFN-γ and LPS, or IL-4 and of IL-13. For glycolysis inhibition, cells were treated with 10mM of 2-Deoxy-D-glucose (2-DG). For inhibition of <i>PKM2</i>, macrophages were treated with 0.25μM of Shikonin. The RNA expressions of proinflammatory cytokines and glycolysis-related genes were detected by qPCR. Immunoblotting, ELISA and confocal miroscopy was applied to examine the expression of <i>PKM2</i>. Extracellular acidification rate (ECAR) analysis was performed to assess glycolysis. The expressions of surface molecule CD80, CD86, and HLA-DR were measured using CytoFLEX flow cytometer. <h3>Results</h3> AS macrophages are prone to produce excessive inflammation, including <i>TNFα</i>, <i>IL1β</i>, and <i>IL23,</i> and are at overactive status by exhibiting stronger co-stimulatory signals, such as CD80, CD86, and HLA-DR. Meanwhile, we found that patient-derived M1 macrophages intensified glycolysis, captured as a higher ECAR. Obviously, upregulation of <i>PKM2</i> and <i>GLUT1</i> has been observed on AS-derived monocytes and macrophages, especially on M1 macrophages, indicating a glucose metabolic alteration in AS macrophages. To investigate how glycolysis impacts macrophage inflammatory ability, 2-DG and Shikonin were applied to AS M1 macrophages, respectively. Consequently, both inhibitors could lessen pro-inflammatory function and reverse overactive status of AS macrophages, potentially favoring disease treatment. <h3>Conclusion</h3> We emphasized hyper-metabolic M1 macrophages of AS and suggested an essential role for PKM2 in inflammatory effector functions of AS macrophages. Targeting PKM2 to inhibit glycolysis in an overactive macrophage may provide novel therapeutic methods for AS inflammation. <h3>Reference</h3> [1]Mauro D, Thomas R, Guggino G, Lories R, Brown MA, Ciccia F. Ankylosing spondylitis: an autoimmune or autoinflammatory disease? Nat Rev Rheumatol. 2021;17(7):387-404. doi:10.1038/s41584-021-00625-y. <h3>Acknowledgements</h3> This work was supported by the Scientific and Technological Planning Project of Guangzhou City [202102020150], Guangdong Provincial Basic and Applied Basic Research Fund Project [2021A1515111172], National Natural Science Foundation of China Youth Fund [82201998], Third Affiliated Hospital of Sun Yat-Sen University Cultivating Special Fund Project for the National Natural Science Foundation of China [2022GZRPYQN01] and National Natural Science Foundation of China [82271849]. <h3>Disclosure of Interests</h3> None Declared.